Scientific Opinion on Flavouring Group Evaluation 91,Revision 3 (FGE.91Rev3): consideration of aliphatic, aromatic and a,b-unsaturated sulfides and thiols evaluated by JECFA(53rd, 61st, 68th and 76th meetings), structurally related to substances in FGE.08

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ADOPTED: 14 May 2020 doi: 10.2903/j.efsa.2020.6154

Scienti ﬁ c Opinion on Flavouring Group Evaluation 91, Revision 3 (FGE.91Rev3): consideration of aliphatic, aromatic

and a , b -unsaturated sul ﬁ des and thiols evaluated by JECFA (53rd, 61st, 68th and 76th meetings), structurally related to

substances in FGE.08Rev5

EFSA Panel on Food Additives and Flavourings (FAF),

Maged Younes, Gabriele Aquilina, Laurence Castle, Karl-Heinz Engel, Paul Fowler, Maria Jose Frutos Fernandez, Peter F€urst, Ursula Gundert-Remy, Rainer G€urtler, Trine Husøy,

Melania Manco, Peter Moldeus, Agneta Oskarsson*, Sabina Passamonti, Romina Shah, Ine Waalkens-Berendsen, Detlef W€olﬂe, Matthew Wright, Romualdo Benigni, Claudia Bolognesi, Kevin Chipman, Eugenia Cordelli, Gisela Degen, Daniel Marzin, Camilla Svendsen, Giorgia Vianello

and Wim Mennes

Abstract

The EFSA Panel on Food Additives and Flavourings was requested to evaluate 49flavouring substances assigned to the Flavouring Group Evaluation 91 (FGE.91), using the Procedure as outlined in the Commission Regulation (EC) No 1565/2000. Forty-four substances have been considered in FGE.91 and its revisions (FGE.91Rev1 and FEG.91Rev2). With regard to the remaining five flavouring substances considered in this revision 3 of FGE.91: two ([FL-no: 12.065 and 12.079]) have been cleared with respect to genotoxicity in FGE.201Rev2; two ([FL-no: 12.169 and 12.241]) were originally allocated to FGE.74Rev4 and one ([FL-no: 12.304]) to FGE.08Rev5. The Panel considered the flavouring substance [FL-no: 12.169] representative for the tertiary monothiols [FL-no: 12.038, 12.085, 12.137, 12.138, 12.145, 12.252, 12.259, 12.241 and 12.304]. The substances were evaluated through a stepwise approach that integrates information on the structure–activity relationships, intake from current uses, toxicological threshold of concern (TTC), and available data on metabolism and toxicity. The Panel concluded that none of these 49 substances gives rise to safety concerns at their levels of dietary intake, estimated on the basis of the ‘Maximised Survey-derived Daily Intake’ (MSDI) approach. The specifications for the materials of commerce have also been considered and found adequate for all 49 flavouring substances. Forfive substances [FL-no: 12.077, 12.162, 12.265, 12.267 and 17.036], evaluated through the Procedure in FGE.91Rev2, no normal and maximum use levels are available. For 10 substances [FL-no: 12.065, 12.038, 12.079, 12.108, 12.139, 12.264, 12.274, 12.252, 12.284 and 12.304], the modified Theoretical Added Maximum Daily Intake (mTAMDI) intake estimates are above the TTC for their structural class. Therefore, for these 15 substances, more detailed data on uses and use levels should be provided in order to refine their exposure assessments and tofinalise their safety evaluations.

Keywords: Flavourings,a,b-unsaturated carbonyls and precursors, FGE.91Rev2, JECFA Requestor:European Commission

* Member of the EFSA Panel on Food Additives and Flavourings (FAF) and of its Working Group on Flavourings until 31 December 2019.

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Question numbers:EFSA-Q-2020-00266, EFSA-Q-2020-00023, EFSA-Q-2020-00022, EFSA-Q-2020-00021, EFSA-Q-2020-00020, EFSA-Q-2020-00019, EFSA-Q-2020-00018, EFSA-Q-2020-00017, EFSA-Q-2020-00016, EFSA-Q-2020-00015, EFSA-Q-2018-00840, EFSA-Q-2018-00839

Correspondence: ﬁ[email protected]

Panel members: Gabriele Aquilina, Laurence Castle, Karl-Heinz Engel, Paul Fowler, Maria Jose Frutos, Fernandez, Peter F€urst, Rainer G G€urtler, Ursula Gundert-Remy, Trine Husøy, Melania Manco, Wim Mennes, Peter Moldeus, Agneta Oskarsson*, Sabina Passamonti, Romina Shah, Ine Waalkens- Berendsen, Detlef W€olﬂe, Matthew Wright and Maged Younes.

Acknowledgments: The Panel wishes to thank the hearing experts Vibe Beltoft and Karin Nørby for the support provided to this scientiﬁc output.

Suggested citation: EFSA FAF Panel (EFSA Panel on Food Additives and Flavourings), Younes M, Aquilina G, Castle L, Engel K-H, Fowler P, Frutos Fernandez P, F€urst P, Gundert-Remy U, G€urtler R, Husøy T, Manco M, Moldeus P, Oskarsson A, Passamonti S, Shah R, Waalkens-Berendsen I, W€olﬂe D, Wright M, Benigni R, Bolognesi C, Chipman K, Cordelli E, Degen G, Marzin D, Svendsen C, Vianello G and Mennes W, 2020. Scientiﬁc Opinion on Flavouring Group Evaluation 91, Revision 3 (FGE.91Rev3):

consideration of aliphatic, aromatic and a,b-unsaturated sulﬁdes and thiols evaluated by JECFA (53rd, 61st, 68th and 76th meetings), structurally related to substances in FGE.08Rev5. EFSA Journal 2020;18(6):6154, 49 pp. https://doi.org/10.2903/j.efsa.2020.6154

ISSN: 1831-4732

This is an open access article under the terms of the Creative Commons Attribution-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited and no modiﬁcations or adaptations are made.

The EFSA Journal is a publication of the European Food Safety Authority, an agency of the European Union.

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1. Introduction

The objective of this revision of FGE.91 (FGE.91Rev3) is to assess:

two a,b-unsaturated substituted sulﬁdes (i.e. [FL-no: 12.065 and 12.079]), cleared with respect to genotoxicity in FGE.201Rev2.¹

The additional genotoxicity, toxicity and exposure data submitted to complete the safety evaluation of 10 tertiary monothiols, represented by ﬂavouring substance [FL-no: 12.169], which were originally allocated to FGE.74Rev4 ([FL-no: 12.169 and 12.241]), FGE.91Rev2 ([FL-no: 12.038, 12.085, 12.137, 12.138, 12.145, 12.252 and 12.259)] and FGE.08Rev5 ([FL-no: 12.304]).

1.1. Background and Terms of Reference as provided by the requestor

1.1.1. Background to Mandate of FGE.201Rev2 (M-2017-0048)

The use of flavouring is regulated under Regulation (EC) No 1334/2008² of the European Parliament and Council of 16 December 2008 on flavourings and certain food ingredients with flavouring properties for use in and on foods. On the basis of Article 9(a) of this Regulation, an evaluation and approval are required for flavouring substances.

The Union List of ﬂavourings and source materials was established by Commission Implementing Regulation (EC) No 872/2012³. The list includes a number of ﬂavouring substances for which the safety evaluation should be completed in accordance with Commission Regulation (EC) No 1565/2000⁴. The substances in this group were included in the Union list with a footnote 1 (under evaluation by EFSA).

In its opinion about this subgroup of 2012, the EFSA Panel considered that the mutagenicity hazard could not be cleared by the endpoints evaluated in the in vivo micronucleus assay submitted. The Panel therefore conclude that further data were required in order to clarify the genotoxic potential of this subgroup. The Panel considered the Comet assay with [FL-no: 05.095] as test material and performed on liver, blood andﬁrst site of contact, as a preferred option to further investigate the genotoxicity in vivo.

The additional data submitted by the applicant consist essentially of:

a transgenic mutation assay in combination with an in vivo micronucleus assay for the substance 2-methylcrotonaldehyde [FL-no: 05.095]

a combination of a Comet/micronucleus assay for the substance 2-methylpent-2-enal [FL-no:

05.090]

The Panel also considered in this opinion on FGE.201 rev.1 that the additional data on 2- methylcrotonaldehyde [FL-no: 05.095] could also be considered representative for the following substances: 2,8-dithianon-4-en-4-carboxaldehyde [FL-no: 12.065] and 2-(methylthiomethyl)but-2-enal [FL-no: 12.079].

1.1.2. Terms of Reference of Mandate from FGE.201Rev2 (M-2017-0048)

The European Commission requests the European Food Safety Authority (EFSA) to evaluate the new information submitted on 2-methylpent-2-enal [FL-no: 05.090] and 2-methylcrotonaldehyde [FL-no:

05.095] including also 2,8-dithianon-4-en-4-carboxaldehyde [FL-no: 12.065] and 2-(methylthiomethyl) but-2-enal [FL-no: 12.079] and, depending on the outcome, proceed to the full evaluation of the substances of this group listed in the table below, in accordance with Commission Regulation (EC) No

1 According to the Mandate and Term of Reference of this FGE, when for aﬂavouring substance the concern for genotoxicity is ruled out, EFSA proceeds to the full evaluation of theseﬂavouring substances, taking into account the requirements of the Commission Regulation (EC) No 1565/2000 and of Regulation (EU) No 1334/2008.

2 Regulation (EC) No 1334/2008 of the European Parliament and of the Council of 16 December 2008 onﬂavourings and certain food ingredients with ﬂavouring properties for use in and on foods and amending Council Regulation (EEC) No 1601/91, Regulations (EC) No 2232/96 and (EC) No 110/2008 and Directive 2000/13/EC. OJ L 354, 31.12.2008, p. 34–50.

3 Commission implementing Regulation (EU) No 872/2012 of 1 October 2012 adopting the list ofﬂavouring substances provided for by Regulation (EC) No 2232/96 of the European Parliament and of the Council, introducing it in Annex I to Regulation (EC) No 1334/2008 of the European Parliament and of the Council and repealing Commission Regulation (EC) No 1565/2000 and Commission Decision 1999/217/EC. OJ L 267, 2.10.2012, p. 1–161.

4 Commission Regulation (EC) No 1565/2000 of 18 July 2000 laying down the measures necessary for the adoption of an evaluation programme in application of Regulation (EC) No 2232/96. OJ L 180, 19.7.2000, p. 8–16.

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1565/2000, within nine months. In case the genotoxic potential cannot be ruled out or the procedure cannot be applied EFSA is asked to characterise the hazards and also quantify the exposure.

As regards the substance 2,6-Dimethly-2,5,7-octatriene-1-ol acetate ([FL-no: 09.931] CAS no 999999-91-4) the applicant indicate that it is included in this subgroup 1.1.2 of FGE19 (FGE.201).

However, this substance has been already evaluated by EFSA in FGE 207 and FGE 72 Rev.1 of 2013.

As regards substance 4-methyl-3-hepten-5-one ([FL-no: 07.261] CAS no 22319-31-9) EFSA indicated in its opinion FGE.204 that ‘the 2-methyl substituted alpha, beta-unsaturated aldehydes in FGE.201Rev1 can be considered as structurally related to it [FL-no: 07.261]. Thus the ﬁnal conclusion on [FL-no: 07.261] will be drawn based on the outcome of the evaluation of FGE.201Rev1’.

1.1.3. Background to Mandate of FGE.91Rev3 joint with FGE.74Rev4 and FGE.08Rev5 (M-2020-0004)

The use of flavourings is regulated under Regulation (EC) No 1334/2008 of the European Parliament and Council of 16 December 2008 on flavourings and certain food ingredients with flavouring properties for use in and on foods. On the basis of Article 9(a) of this Regulation, an evaluation and approval are required for flavouring substances.

The Union list of flavourings and source materials was established by Commission Implementing Regulation (EC) No 872/2012. The list contains flavouring substances for which the scientific evaluation should be completed in accordance with Commission Regulation (EC) No 1565/2000 and taking into account also the provisions of Regulation (EC) No 1334/2008.

Additional information was submitted regarding the group of substances belonging to the Flavouring Group Evaluation 74. In January the EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids adopted the opinion on the Flavouring Group Evaluation 74 Revision 4, (FGE.74Rev4)⁵ and concluded that for the two tertiary thiols [FL-no: 12.169 and 12.241] further data are required. The Panel did not agree with JECFA that appropriate studies were available for deriving NOAELs for these two tertiary thiols. Therefore, the evaluation could not be considered completed for these substances.

On December 2017 the applicant submitted additional information consisting of a 90-day study (on substance [FL-no: 12.169]) and also poundage and usage information concerning substances from FGE.74 and FGE.91. As EFSA is currently evaluating two newly included substances in the group FGE.91 revision 3, not considered in earlier revisions of this FGE, it is now appropriate to also consider this additional information submitted on the substances represented by 2-methyl-4-oxopentane-2-thiol [FL-no: 12.169], in order toﬁnalize their safety assessment through the Procedure.

The current request also concerns theﬂavouring substance [FL-no: 12.304]. This substance [FL-no:

12.304] is structurally related to the other 9 tertiary monothiols in FGE.91Rev3 and FGE.74Rev4 and therefore can be represented by 2-methyl-4-oxopentane-2-thiol [FL-no: 12.169]. The substance [FL- no: 12.304] is currently included in the FGE.08Rev5.⁶ FGE.08Rev5 is a supporting FGE both for FGE.91 and FGE.74. FGE.08 includes non-JECFA-evaluated substances structurally related to the ones in FGE.91 and 74. The safety evaluation through the Procedure of four substances included in FGE.08Rev5 with [FL-no: 12.304, 12.172, 12.174 and 16.057], was not concluded as the assessment of their genotoxicity potential could not be carried out at the time of the ﬁnalization of revision 5 of FGE.08. However, [FL-no: 12.172, 12.174 and 16.057] were no longer supported by industry and they were not in included in the Union List of ﬂavourings. Therefore, the only safety evaluation through the Procedure which is still pending for the substances in FGE.08Rev5 is the one for the remaining substance [FL-no: 12.304]. The concern for genotoxicity for this substance could be addressed by considering the genotoxicity data made available on the representative substance for the tertiary monothiols, i.e. [FL-no: 12.169], mentioned in the initial request. In addition, since [FL-no: 12.304]

was evaluated by JECFA in 2012, its safety assessment could be adequately ﬁnalized in FGE.91Rev3 since now [FL-no: 12.304] is a JECFA-evaluated substance (FGE.50 up to FGE.99 contain only JECFA- evaluated substances).

5 Scientiﬁc Opinion on Flavouring Group Evaluation 74, Revision 4 (FGE.74Rev4): Consideration of Simple Aliphatic Sulphides and Thiols evaluated by JECFA (53rd and 61st meeting) Structurally related to Aliphatic and Alicyclic Mono-, Di-, Tri-, and Polysulphides with or without Additional Oxygenated Functional Groups from Chemical Group 20 evaluated by EFSA in FGE.08Rev5 (2012). EFSA Journal 2018;16(3):5167, 58 pp.https://doi.org/10.2903/j.efsa.2018.5167

6 Scientiﬁc Opinion on Flavouring Group Evaluation 08, Revision 5 (FGE.08Rev5): Aliphatic and alicyclic mono-, di-, tri-, and polysulphides with or without additional oxygenated functional groups from chemical groups 20 and 30. https://doi.org/10.

2903/j.efsa.2012.2837. Available online:www.efsa.europa.eu/efsajournal

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1.1.4. Terms of Reference of FGE.91Rev3 joint with FGE.74Rev4 and FGE.08Rev5 (M-2020-0004)

The European Commission requests the European Food Safety Authority (EFSA) to evaluate this new information and to proceed with the full evaluation of these ﬂavouring substances in accordance with Commission Regulation (EC) No 1565/2000 and taking into account also Regulation (EC) No 1334/2008. The assessment should be carried out within 6 months from the receipt of this letter, e.g.

up to 30th June 2020.

1.2. Interpretation of the Terms of Reference

The Panel considered that for the two sulﬁdes [FL-no 12.065 and 12.079] the evaluation of their possible genotoxic properties in FGE.201Rev2 was justiﬁed considering the structural similarity, with respect to thea,b-unsaturated carbonyl moiety, to the other substances within FGE.201. Since the concern for genotoxicity has been alleviated in FGE.201Rev2, the assessment can proceed in the current revision of FGE.91 as the substances in this group are structurally similar with respect to the sulfur function.

In a previous submission, industry also provided an additional in vitro genotoxicity assay on ﬂavouring substance [FL-no: 12.169] (Documentation provided to EFSA nr: 6). This assay was not considered in FGE.74Rev4 where the genotoxicity potential of this substance, and its other structurally related substances, was initially investigated and ruled out. Since this ﬂavouring substance [FL-no:

12.169] will be included in the current revision of FGE.91, this study will now be included and assessed in this revision.

1.2.1. History of the evaluation of the substances in Flavouring Group Evaluation 91

The EFSA consideration in FGE.91 (EFSA CEF Panel, 2010) dealt with 45 substances, 40 simple aliphatic and aromatic sulﬁdes and thiols evaluated by JECFA at the 68th meeting (JECFA, 2007) and ﬁve tertiary thiols evaluated by JECFA at the 53rd meeting (JECFA, 2000). For seven tertiary mono thiols [FL-no: 12.038, 12.085, 12.137, 12.138, 12.145, 12.252 and 12.259], it was concluded that adequate genotoxicity data were needed before the substances could be evaluated through the Procedure.

Thefirst revision of FGE.91, FGE.91Rev1 (EFSA CEF Panel, 2011a), included the assessment of two additional substances, benzyl methyl sulfide [FL-no: 12.077] and methyl phenyl sulfide [FL-no: 12.162]

(in total 47 substances). These substances were evaluated by JECFA at its 53rd meeting (JECFA, 2000).

Since publication of FGE.91Rev1, additional genotoxicity data have become available for 4- mercapto-4-methyl-2-pentanone [FL-no: 12.169] from FGE.74Rev3 (EFSA CEF Panel, 2014b). This substance was considered representative for the seven tertiary monothiols [FL-no: 12.038, 12.085, 12.137, 12.138, 12.145, 12.252 and 12.259] for which a concern with respect to genotoxicity was identiﬁed in FGE.91.

After the publication of FGE.91Rev1, three (i.e. [FL-no: 12.114, 12.256 and 12.272]) candidate substances were no longer supported by the industry for use as flavouring substances in Europe⁷and therefore they were not considered any further. The second revision of FGE.91 (EFSA CEF Panel, 2014a), FGE.91Rev2 (in total including 44 substances), concerned the assessment of the newly submitted genotoxicity data on [FL-no: 12.169] used to cover the seven tertiary monothiols. The available data were limited but the CEF Panel considered that these genotoxicity data did not preclude the evaluation of the substances in FGE.91Rev2 through the Procedure. In FGE.91Rev2, the Panel concluded that for the tertiary mono thiols [FL-no: 12.038, 12.085, 12.137, 12.138, 12.145, 12.252 and 12.259], contrary to the JECFA, there are no adequate no observed adverse effect levels (NOAELs) and that additional toxicity data are required to finalise the evaluation of these seven substances. In addition, the modified Theoretical Added Maximum Daily Intake (mTAMDI) figures for five substances [FL-no: 12.264, 12.284, 12.274, 12.108 and 12.139] were above the threshold of concern for their structural classes. For these substances more detailed data on uses and use levels are needed. For 10 substances [FL-no: 12.038, 12.077, 12.085, 12.137, 12.138, 12.145, 12.162, 12.265, 12.267 and 17.036], no use levels have been provided.

7 DG SANCO (Directorate General for Health and Consumer Affairs), 2013a. Information from DG SANCO 14/05 2013, concerning a list of 18 non-supported substances. FLAVIS.2.26.

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The present revision of FGE.91, FGE.91Rev3, includes the safety evaluation of two additional sulfides: 2-(methylthiomethyl)but-2-enal [FL-no: 12.079] and 2,8-dithianon-4-en-4-carboxaldehyde [FL- no: 12.065]. These substances were evaluated by JECFA in its 53rd meeting (JECFA, 2000) and cleared with respect to genotoxicity in FGE.201Rev2 (EFSA CEF Panel, 2018b). By expert judgement, these substances have been included in the current revision 3 of FGE.91 on the basis of their structural similarity with the substances considered in this FGE. The present revision includes also the completion of the safety evaluation of seven tertiary monothiols [FL-no: 12.038, 12.085, 12.137, 12.138, 12.145, 12.252 and 12.259], which were pending from the previous revision (FGE.91Rev2) owing to lack of toxicological data (i.e. a 90-day study to identify a NOAEL). A 90-day toxicity study performed on 4- mercapto-4-methyl-2-pentanone [FL-no: 12.169] (from FGE.74Rev4, EFSA CEF Panel, 2018a), chosen as representative substance to cover the evaluation of tertiary monothiols in FGE.74 ([FL-no: 12.169 and 12.241]) and FGE.91 ([FL-no: 12.038, 12.085, 12.137, 12.138, 12.145, 12.252 and 12.259]), has been provided and a NOAEL can be identified. The Panel considers that the chemical structures of the two substances from FGE.74Rev4 are sufficiently structurally related to those of the tertiary monothiols in FGE.91 and therefore agrees to include these two substances in FGE.91Rev3. New poundage and use levels data have been submitted for these nine substances [FL-no: 12.169, 12.241, 12.038, 12.085, 12.137, 12.138, 12.145, 12.252 and 12.259] and these new exposure data will be included in the present opinion (Documentation provided to EFSA nr: 4). In addition, this revision 3 of FGE.91 includes the finalisation of the safety evaluation of substance ethyl-2-mercapto-2-methyl propanoate [FL-no: 12.304] which was originally allocated in FGE.08Rev5 (EFSA CEF Panel, 2012a). The safety evaluation through the Procedure of this flavouring substance was not concluded in FGE.08Rev5 as the genotoxicity concern could not be ruled out due to lack of data. The concern for genotoxicity for this substance could now be ruled out by the genotoxicity data available on the representative substance for the tertiary monothiols, i.e. [FL-no: 12.169]. Moreover, since [FL-no: 12.304] had been evaluated by JECFA at its 76th meeting (JECFA-no: 2085, JECFA, 2012), the Panel agrees to finalise its safety assessment directly in FGE.91Rev3 as now it is a JECFA-evaluated substance and it is structurally related to the other nine tertiary monothiols in FGE.91Rev3.

Therefore, together with the 44 substances that were already considered in FGE.91Rev2 and the ﬁve newly included substances, i.e. [FL-no: 12.169 and 12.241] from FGE.74Rev4, [FL-no: 12.079 and 12.065] from FGE.201Rev2 and [FL-no: 12.304] from FGE.08Rev5, the current revision comprises 49 substances. The Panel agrees that all 49 JECFA-substances in FGE.91Rev3 are structurally related to the aliphatic and alicyclic mono-, di-, tri- and polysulﬁdes with or without additional oxygenated functional groups evaluated by EFSA in the FGE.08Rev5 (EFSA CEF Panel, 2012a).

The 44ﬂavouring substances for which the evaluation was ﬁnalised in FGE.91Rev2 will not further be discussed. Nevertheless, for the sake of completeness their information is maintained in the various tables in this FGE.

FGE Adopted by EFSA Link No. of

substances FGE.91 24 September 2009 https://www.efsa.europa.eu/en/efsajournal/pub/688 45 FGE.91Rev1 23 November 2011 https://www.efsa.europa.eu/en/efsajournal/pub/1026 47 FGE.91Rev2 21 May 2014 https://www.efsa.europa.eu/en/efsajournal/pub/3707 44 FGE.91Rev3 14 May 2020 https://www.efsa.europa.eu/en/efsajournal/pub/6154 49

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2. Data and methodologies

2.1. Data

The present revision of the opinion on FGE.91 is based on the following data as provided by the applicant:

FL-no Chemical name

Data provided for the current revision 3 of FGE.91

Appendix (Table nr)/

relevant section of the opinion

Documentation provided to EFSA/Reference 12.065 2,8-Dithianon-4-en-4-

carboxaldehyde

Speciﬁcations, EU poundage data (MSDI), use levels (mTAMDI)

AppendixB(TableB.1), AppendixC(TablesC.1 andC.4)

Documentation provided to EFSA nr: 1, 2, 3 12.079 2-(Methylthiomethyl)-

but-2-enal

12.038 8-Mercapto-p-menthan- 3-one

EU poundage data (MSDI), use levels (mTAMDI)

AppendixC(TablesC.1 andC.4)

Documentation provided to EFSA nr: 4

12.085 p-Menth-1-ene-8-thiol 12.137 3-Mercapto-3-

methylbutan-1-ol 12.138 3-Mercapto-3-

methylbutyl formate 12.145 4-Methoxy-2-

methylbutane-2-thiol 12.252 4-Mercapto-4-methyl-2-

pentanol

12.259 1-Mercapto-p-menthan- 3-one

12.241 2-Mercapto-2- methylpentan-1-ol 12.169 2-Methyl-4-oxopentane-

2-thiol

EU poundage data (MSDI), use levels (mTAMDI), Genotoxicity and toxicity data

AppendixC(TablesC.1 andC.4), Section3.3.2.1.

AppendixE(TableE.1)

Documentation provided to EFSA nr: 4, 5, 6 FL-no: FLAVIS number; MSDI: Maximised Survey-derived Daily Intake; mTAMDI: modiﬁed Theoretical Added Maximum.

In addition, the following references were used:

JECFA speciﬁcations for the two newly allocated ﬂavouring substances [FL-no: 12.065 and 12.079] (JECFA, 2003, 2005).

53rd, 61st and 76th JECFA reports (JECFA, 2000, 2004, 2012).

Genotoxicity data evaluated in FGE.201Rev2 (EFSA CEF Panel, 2018b).

EFSA scientiﬁc opinions on FGE.91 and its revisions (EFSA CEF Panel, 2010, 2011a, 2014).

EFSA scientiﬁc opinion on FGE.74Rev4 (EFSA CEF Panel, 2018a).

EFSA scientiﬁc opinion on FGE.08Rev5 (EFSA CEF Panel, 2012a).

2.2. Methodologies

This opinion follows the principles described in the EFSA Guidance on transparency with regard to scientific aspects of risk assessment (EFSA Scientific Committee, 2009) and the relevant existing guidance documents from the EFSA Scientific Committee. The assessment strategy applied for the evaluation programme offlavouring substances, as laid down in Commission Regulation (EC) No 1565/

2000, is based on the Opinion on a Programme for the Evaluation of Flavouring substances of the Scientiﬁc Committee on Food (SCF, 1999).

2.2.1. Procedure for the safety evaluation of ﬂavouring substances

The approach for safety evaluation of chemically deﬁned ﬂavouring substances as referred to in Commission Regulation (EC) No 1565/2000, named the‘Procedure’, is described in AppendixA.

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2.2.2. Approach used for the calculation of exposure

The approach used for calculation of the intake of the ﬂavouring substances is described in Appendix A(point‘a)Intake’) and in AppendixC(Section C.2‘mTAMDI calculation’).

3. Assessment

3.1. Speci ﬁ cations

JECFA specifications are available for all 49 flavouring substances in FGE.91Rev3 (JECFA, 2003, 2005, 2012). Allflavouring substances are shown in Table B.1–Appendix B.

EFSA considerations

Table1 shows the chemical structures of the substances considered in this revision of FGE.91 (FGE.91Rev3).

Table 1: Flavouring substances under evaluation in FGE.91Rev3.

FL-no Chemical structure Chemical name Structural class*

12.085 p-Menth-1-ene-8-thiol I

12.169 2-Methyl-4-oxopentane-2-thiol

(4-mercapto-

4-methyl-2-pentanone)

III

12.241 2-Mercapto-2-methylpentan-1-ol III

12.137 3-Mercapto-3-methylbutan-1-ol III

12.138 3-Mercapto-3-methylbutyl formate III

12.145 4-Methoxy-2-methylbutane-2-thiol III

12.252 4-Mercapto-4-methyl-2-pentanol III

12.259 1-Mercapto-p-menthan-3-one III

12.038 8-Mercapto-p-menthan-3-one III

12.304 Ethyl-2-mercapto-2-methyl propanoate III

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The two newly allocated substances [FL-no: 12.065 and 12.079] are a,b-unsaturated substituted aldehydes and they can exist as geometrical stereoisomers. The applicant provided adequate information with respect to the composition of the stereoisomeric mixtures (see Table B.1 – Appendix B) (Documentation provided to EFSA nr: 1). For the remaining candidate substances in FGE.91Rev3, the speciﬁcations were considered in FGE.74Rev4 ([FL-no: 12.169 and 12.241]), FGE.91Rev2 ([FL-no: 12.038, 12.085, 12.137, 12.138, 12.145, 12.252 and 12.259]) and FGE.08Rev5 ([FL-no: 12.304]) and found adequate (EFSA CEF Panel, 2012a, 2014a, 2018a).

According to the information provided by industry, ﬂavouring substance [FL-no: 12.169] is supplied as 1% solution in propylene glycol of a mixture of 2-methyl-4-oxopentane-2-thiol (48%) and 4-methyl- 3-penten-2-one (48–50%) (EFSA CEF Panel, 2018a). The Panel noted that the latter is an authorised ﬂavouring substance ([FL-no: 07.101]). Moreover, as already indicated in FGE.74Rev4, the chemical name 2-methyl-4-oxopentane-2-thiol should be changed to4-mercapto-4-methyl-2-pentanone.

The most recent speciﬁcations data for all substances in FGE.91Rev3 are summarised in Table B.1– Appendix B.

3.2. Estimation of intake

JECFA status

For all 49ﬂavouring substances, evaluated through the JECFA Procedure, intake data are available for the EU (JECFA, 2000, 2004, 2012).

EFSA considerations

EU productionﬁgures for allﬂavouring substances under evaluation in FGE.91Rev3 ([FL-no: 12.065, 12.079, 12.038, 12.085, 12.137, 12.138, 12.145, 12.252, 12.259, 12.169, 12.241 and 12.304]) have been provided by industry (EFSA CEF Panel 2012a, 2014a, 2018a; Documentation provided to EFSA nr: 2, 3, 4) and MSDI values have been calculated ranging from 0.012 to 37 lg/capita per day.

Normal and maximum use levels have been provided for flavouring substances [FL-no: 12.065, 12.079, 12.038, 12.085, 12.137, 12.138, 12.145, 12.252, 12.259, 12.169, 12.241 and 12.304], (Documentation provided to EFSA n. 3 and 4) and mTAMDI intake values have been calculated. The mTAMDI intake estimate calculated from these data for flavouring substance [FL-no: 12.085] is below the toxicological threshold of concern (TTC) for its structural class I. The mTAMDI intake estimates for flavouring substances [FL-no: 12.137, 12.138, 12.145, 12.169 and 12.241] are below the TTC for their structural class III. The mTAMDI intake estimates for flavouring substances [FL-no: 12.065, 12.079, 12.304, 12.038 and 12.252] are above the TTC for their structural class III. From the previous revision (FGE.91Rev2) the mTAMDI intake values for flavouring substances [FL-no: 12.264, 12.284, 12.274, 12.108 and 12.139] are above the TTC for their structural classes. Therefore, for these 10 substances ([FL-no: 12.065, 12.079, 12.304, 12.038, 12.252, 12.264, 12.284, 12.274, 12.108 and 12.139]), more detailed data on uses and uses levels should be provided in order to refine the exposure assessment and to finalise their safety evaluations.

No normal and maximum use levels have been provided for ﬂavouring substances [FL-no: 12.077, 12.162, 12.265, 12.267 and 17.036], previously evaluated in FGE.91Rev2.

The MSDI values and the mTAMDI intake estimates for substances in FGE.91Rev3 are shown in Table C.4 –Appendix C.

FL-no Chemical structure Chemical name Structural class*

12.065 2,8-Dithianon-4-en-4- carboxaldehyde III

12.079 2-(Methylthiomethyl)-but-2-enal III

FL-no: FLAVIS number.

*: Determined with OECD Toolbox (version 4.3.1 available athttps://www.oecd.org/chemicalsafety/risk-assessment/oecd-qsar- toolbox.htm).

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3.3. Biological and toxicological data

3.3.1. ADME data

The two candidate substances [FL-no: 12.065 and 12.078] area,b-unsaturated sulfides which were evaluated by JECFA at its 53rd meeting, within a group of 137 sulfur-containing substances, particularly in the ‘subgroup ii acyclic sulfides with oxidised side-chains’ (JECFA, 2000). In the 53rd JECFA report (JECFA, 2000), these substances are described as sufficiently lipophilic to be absorbed from the intestine and they would be metabolised via many different pathways. The presence of other functional groups, such the a,b-unsaturated aldehydes in the two candidate substances, provides greater polarity and an additional site for the biotransformation of these sulfides. According to JECFA, the presence of these polar groups would also enhance the renal excretion (JECFA, 2000). Concurrent metabolic pathways of various sulfides substituted with oxygenated functions were reported, but sulfoxide formation, via enzymatic catalysis (P450 and flavin-containing monooxygenases), is the predominant metabolic pathway of detoxification. Sulfoxides may be further oxidised to sulfones and these two are the main urinary metabolites of sulfides.

The other 10 candidate substances are tertiary monothiols. Seven of them were evaluated in the 53rd JECFA meeting within the ‘subgroup v thiols with oxidised side-chains’ (JECFA, 2000) ([FL-no:

12.038, 12.085, 12.137, 12.138, 12.145, 12.252 and 12.259]), two substances at the 61st JECFA meeting (JECFA, 2004) ([FL-no: 12.169 and 12.241]) and one substance ([FL-no: 12.304]) at the 76th JECFA meeting (JECFA, 2012). Substances both from 61st and 76th JECFA meetings were evaluated within the same subgroup v of the original group of 137 sulfur-containing substances, previously evaluated at the 53rd JECFA meeting. According to JECFA, the metabolism of thiols with oxidised side- chains is predicted to involve a combination of pathways for simple thiols together with further oxidation or conjugation of the oxidised side-chain. Metabolic options for thiols in mammals are thiol methyltransferase catalysed S-methylation to yield the corresponding thioethers and sulfides, which are then oxidised to methyl sulfoxides and methyl sulfones. S-adenosyl-L-methionine is required as a methyl group donator. Simple thiols may undergo oxidation to form unstable sulfenic acids (RSOH) which are oxidised to sulfinic acids (RSO₂H) and then to sulfonic acids (RSO₃H) or combined with nucleophilic sites. Thiols may also react with glutathione (GSH) and other endogenous thiols to generate disulfides via bio-catalysis with microsomal and cytosolic thioltransferases. The disulfides can be reduced back to thiols, oxidatively desulfurated or oxidised to sulfonic acid.

JECFA could not conclude that the expected resulting metabolites are innocuous and evaluated the 12 candidate substances [FL-no: 12.065, 12.078, 12.169, 12.241, 12.038, 12.085, 12.137, 12.138, 12.145, 12.252, 12.259 and 12.304] along the B-side of the Procedure scheme.

EFSA considerations

Regarding the two oxygenated sulfides [FL-no: 12.065 and 12.079], the Panel observed that one of the candidate flavouring substance, i.e. [FL-no: 12.065], was used as a supporting substance in the evaluation of the acyclic sulfides subgroup in FGE.08Rev5 (EFSA CEF Panel, 2012a). In FGE.08Rev5 (EFSA CEF Panel, 2012a), there is an extensive description of the possible metabolic routes for the acyclic sulfides, which are in line with those outlined by JECFA. These substances are sufficiently lipophilic to be absorbed and they rapidly undergo oxidation catalysed by monooxygenase systems (P450 and FMO) to yield sulfoxides (S-oxidation). Sulfoxides may be further transformed to sulfones via an irreversible oxidation catalysed by P450. However, the sulfoxide is generally the predominant urinary metabolite of simple sulfides, such as methyl sulfide. When a sulfide contains also an oxygenated functional group, like the two candidate substances [FL-no: 12.065 and 12.078], for the oxygenated functional group the common biotransformations of alcohol, acid and carbonyl functional groups (C-oxidation and/or conjugation) would be expected. For oxygenated sulfides, C-oxidation and/

or conjugation may compete with S-oxidation, nevertheless sulfoxide formation is usually the major metabolic pathway.

With regard to the 10 tertiary monothiols, under evaluation in FGE.91Rev3 ([FL-no: 12.169, 12.241, 12.038, 12.085, 12.137, 12.138, 12.145, 12.252, 12.259 and 12.304]), some of them were used as supporting substances in the evaluation of the thiols subgroup in FGE.08Rev5 (EFSA CEF Panel, 2012a) and [FL-no: 12.304] was originally allocated to FGE.08Rev5. According to FGE.08Rev5, and in line with JECFA evaluations, these substances would be expected to be detoxiﬁed via a combination of pathways including S-oxidation, methylation, oxidative desulfuration, alkylation, and conjugation with

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GSH and/or glucuronic acid. The electrophilic metabolites, resulting from the S-oxidation and/or other reactions (i.e. oxidative desulfuration, alkylation, hydrolysis), can react with endogenous thiols present in cellular macromolecules (such as cysteine or reduced GSH) or with other nucleophilic cellular sites leading to the formation of perthiols. These substances are strong reductants and can interact with free radicals to produce perthiyl radicals which undergo prooxidative reactions.

Therefore, based on the reactivity of the expected metabolites, the Panel agrees to evaluate all the 12 candidate substances [FL-no: 12.065, 12.079, 12.169, 12.241, 12.038, 12.085, 12.137, 12.138, 12.145, 12.252, 12.259 and 12.304] via the B-side of the Procedure scheme (see Appendix A).

3.3.2. Genotoxicity data

This revision involves the inclusion of two flavouring substances [FL-no: 12.065 and 12.079], for which in FGE.19 a concern for genotoxicity had been identified based on the presence of a structural alert (i.e. a,b-unsaturated carbonyl substance or precursor for that), precluding their evaluation through the Procedure (EFSA CEF Panel, 2011b). Therefore, these substances needed further attention in FGE.201 and its revision 2 (FGE.201Rev2), where their genotoxic potential has been assessed and the concern for genotoxicity was ruled out (EFSA CEF Panel, 2018b). Therefore, the safety evaluation through the Procedure can be performed for theseflavouring substances [FL-no: 12.065 and 12.079].

FGE.91Rev3 also deals with thefinalisation of the safety evaluation through the Procedure for nine tertiary monothiols [FL-no: 12.169, 12.241, 12.038, 12.085, 12.137, 12.138, 12.145, 12.252 and 12.259]. The genotoxicity of these substances was investigated and the concern for the genotoxicity was ruled out in FGE.91Rev2 and FGE.74Rev4 (EFSA CEF Panel, 2014a, 2018a) based on genotoxicity data on the representative substance 2-methyl-4-oxopentane-2-thiol (4-mercapto-4-methyl-2- pentanone) [FL-no: 12.169] and other genotoxicity data (from JECFA and FGE.08Rev5) on structurally related substances. With regard to the flavouring substance [FL-no: 12.304], originally allocated in FGE.08Rev5 (EFSA CEF Panel, 2012a), its safety evaluation could not befinalised in FGE.08Rev5 as the genotoxicity concern was not ruled out. The concern for genotoxicity of [FL-no: 12.304] can now be ruled out based on the available genotoxicity data on the representative substance 2-methyl-4- oxopentane-2-thiol (4-mercapto-4-methyl-2-pentanone) and thus [FL-no: 12.304] can be evaluated through the Procedure in this FGE.

3.3.2.1. In vitromicronucleus assay on human peripheral blood lymphocytes on 2-methyl-4-oxopentane-2-thiol (4-mercapto-4-methyl-2-pentanone) (Documentation provided to EFSA nr: 6)

The ﬂavouring substance 2-methyl-4-oxopentane-2-thiol (4-mercapto-4-methyl-2-pentanone) [FL- no: 12.169] (purity 99.9%) in dimethyl sulfoxide (DMSO) solution was tested in an in vitro micronucleus (MN) assay using duplicate human lymphocyte cultures in a single experiment consisting ofﬁve separate trials, as in accordance to OECD guideline 487. The vehicle control was DMSO solution.

Mitomycin C (MMC) and Vinblastine (VIN) were used as clastogenic and aneugenic positive control, respectively.

Based on the results of a range-ﬁnding test, cultures were treated up to 1,323lg/mL, equivalent to 10 mM, for 3 + 21 h in the absence and presence of S-9 and up to a cytotoxic concentration (90.00 lg/mL) for 24 + 0 h in the absence of S-9. The test article concentrations for MN analysis were selected by evaluating the effect of 2-methyl-4-oxopentane-2-thiol on the replication index. Cultures from all three treatment conditions were analysed for micronuclei at four concentrations in 2,000 binucleated per concentration cells.

From the study results, the Panel concludes that 2-methyl-4-oxopentane-2-thiol (4-mercapto-4- methyl-2-pentanone) [FL-no: 12.169] did not induce biologically relevant increases in the frequency of micronuclei in cultured human peripheral blood lymphocytes under the tested conditions. This further supports the conclusions on genotoxicity previously reached in FGE.74Rev4 (EFSA CEF Panel, 2018a) and conﬁrms thatﬂavouring substance [FL-no: 12.169], and its structurally related substances [FL-no:

12.038, 12.085, 12.137, 12.138, 12.145, 12.241, 12.252, 12.259 and 12.304], do not pose a concern with respect to genotoxicity.

3.3.3. Toxicological data

One subacute toxicity study is available for candidate substance [FL-no: 12.065] (Central Institute for Nutrition and Food Research; 1974, see Appendix E). This study is not suitable for the assessment of the two sulﬁdes candidate substances ([FL-no: 12.065 and 12.079]) because of the too short

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duration. In FGE.08Rev5, a subchronic toxicity study is available for dimethyl sulﬁde [FL-no: 12.006].

This study was used for the evaluation of substances in FGE.08Rev5 (acyclic sulﬁdes) that are structurally related to the two sulﬁdes candidate substances in FGE.91Rev3.

Forﬂavouring substance 2-Methyl-4-oxopentane-2-thiol (4-mercapto-4-methyl-2-pentanone) [FL-no:

12.169], selected as representative substance for the tertiary monothiols thiols in FGE.91Rev3 ([FL-no:

12.241, 12.038, 12.085, 12.137, 12.138, 12.145, 12.252 and 12.259 and 12.304]), industry submitted toxicological studies which are described below (Documentation provided to EFSA nr: 5).

All the available toxicity studies are summarised in TableE.1of Appendix E.

3.3.3.1. Subchronic toxicity study on dimethyl sulﬁde [FL-no: 12.006] (FGE.08Rev5, EFSA CEF Panel, 2012a)

Four groups of 15 Wistar rats per sex were given dimethyl sulfide by daily gavage in corn oil at dose levels of 2.5, 25 or 250 mg/kg body weight (bw) for 14 weeks. The control group received the same volume of corn oil only. Additional two groups (five/sex per dose) were given daily doses of 0.25 or 250 mg/kg bw for 2 or 6 weeks, respectively. The animals were weighed on day 0 and then weekly throughout the study. Food and water consumption were measured over a 24-h period preceding the day of weighing. Urine samples were collected during weeks 2, 6 and 14, and examined for volume, appearance, specific gravity, microscopic constituents, and content of glucose, ketones, bile salts and blood. At sacrifice, blood was taken for haematological examinations. Gross abnormalities and organ weights were recorded. Histological examinations were also performed. There was no adverse effect at any level in treated rats, and therefore, 250 mg/kg bw per day, the highest dose tested, was considered as the NOAEL.

3.3.3.2. Toxicological studies on 2-methyl-4-oxopentane-2-thiol (4-mercapto-4-methyl-2- pentanone) (Documentation provided to EFSA nr: 5)

Repeated dose range-ﬁnding study

Forty adult Crl: Sprague–Dawley CD^® IGS rats (20 males and 20 females) were equally distributed into four Groups (5/sex per group). Dose levels of 15, 65 and 130 lg/kg bw per day of 2-methyl-4 oxopentane-2-thiol as well as a vehicle control (propylene glycol) were administered via gavage for 14 days.

Cage side observations were recorded daily for all animals and a battery of detailed clinical observations was performed once weekly. Body weights and food consumption were collected once weekly and body weight gain and food efﬁciency were calculated.

There were no mortalities or clinical effects. Investigations of body weight, body weight gain, food consumption, or food efﬁciency did not reveal any changes attributable to 2-methyl-4-oxopentane-2- thiol administration.

There were no macroscopic changes attributable to 2-methyl-4-oxopentane-2-thiol administration.

Under the conditions of this study and based on the toxicological endpoints evaluated, male and female rats are expected to tolerate dose levels of 130lg/kg bw per day in a study of longer duration.

Subchronic toxicity study

Sprague–Dawley CD^® IGS rats (10/sex per group) received by gavage a 1% solution of 2-methyl-4- oxopentane-2-thiol in propylene glycol, resulting in nominal dose levels of 0 (control, 10% propylene glycol, 10 mL/kg bw per day), 130, 200 and 260lg/kg bw per day for 90 consecutive days.

The stability of the test material, 2-methyl-4-oxopentane-2-thiol, was investigated via head space gas chromatography–mass spectrometry. The nominal dose levels mentioned above reﬂect the actual exposure in the different dose groups.

The study was conducted in compliance with OECD guideline 408 (from 1998) which was applicable when the study was conducted. The Panel noted that the highest dose levels did not induce toxicity as required by this OECD guideline. However, the strong odour of the tested substance precluded inclusion of higher doses in the study design, which would have resulted in a wider dose range.

All animals survived the treatment period. No treatment-related clinical signs were observed. There were no treatment-related changes in haematology, coagulation or urinalysis parameters. At the highest dose tested (260 lg/kg bw per day) on day 44, a slight increase in cholesterol was observed in females. However, this clinical chemistry change was not considered adverse and it was not correlated to any histopathology ﬁndings. Pathology and histopathology revealed no increase in the

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absolute and relative organ weights. There were no treatment-related macroscopic or microscopic ﬁndings in any of the treated rats examined.

In an additional report for the same study, the inﬂuence of the test substance on reproduction function parameters (including oestrus cyclicity and sperm motility, epididymal sperm count, homogenisation-resistant spermatid count and morphology) in male and females was described. None of these study parameters was affected by the exposure to the test substance.

The NOAEL for the 90-day study was the highest dose administered, i.e. 260lg/kg bw per day of 2-methyl-4-oxopentane-2-thiol. The Panel agrees with the conclusions of the study report.

The Panel noted that, according to the information provided in the study report, the test item in the repeated dose toxicity study was a 1% solution of 2-methyl-4-oxopentane-2-thiol in propylene glycol that did not contain 4-methyl-3-penten-2-one [FL-no: 07.101], which is a secondary component in the material of commerce of [FL-no: 12.169].

EFSA Considerations

Regarding the available subchronic toxicity study for dimethyl sulfide [FL-no: 12.006] available in FGE.08Rev5, the Panel noted that this compound has been used as a representative substance to cover the evaluation of the subgroup of acyclic sulfides, with and without oxygenated functional groups, in FGE.08Rev5 (EFSA CEF Panel, 2012a). The Panel considered that the two sulfides [FL-no:

12.065 and 12.079], according to their chemical structures, can be considered structurally related to the substances in this subgroup. In addition, based on the available information on ADME (see Section 3.3.1), the expected metabolic pathways involved would be the same for all the acyclic sulfides (different chain length and with or without oxygenated functional groups). Therefore, the Panel agreed that the NOAEL (250 mg/kg bw per day) for dimethyl sulfide [FL-no: 12.006] can be used to derive a margin of safety for the two sulfides [FL-no: 12.065 and 12.079].

3.4. Application of the Procedure

Application of the Procedure to two substances from subgroup ii – acyclic sulﬁdes with oxidised side-chains and 10 substances from subgroup v – thiols with oxidised side-chains (JECFA, 2000, 2004, 2012).

Subgroup ii– acyclic sulﬁdes with oxidised side-chains

In the 53rd JECFA meeting report, the two sulﬁdes [FL-no: 12.065 and 12.079], under evaluation in FGE.91Rev3, were allocated to structural class I, according to Cramer et al. (1978).

JECFA considered that these two ﬂavouring substances cannot be anticipated to be metabolised to innocuous products and accordingly they should be evaluated along the B-side of the Procedure scheme. JECFA considered the available NO(A)ELs for structurally substances 2-(methylthiomethyl)-3- phenylpropenal (JECFA-no: 505) and dimethyl sulﬁde (JECFA-no: 452, [FL-no: 12.006]) inappropriate for evaluating the toxicity of the two candidate substances [FL-no: 12.065 and 12.079], since JECFA expected these substances ([FL-no: 12.065 and 12.079]) to be potentially more reactive and toxic.

Therefore, the JECFA evaluation proceeded to step B5 of their Procedure where JECFA concluded that the substances [FL no: 12.065 and 12.079] do not raise a safety concern because their exposure estimates (based on MSDIs) were below the threshold of 1.5lg/person per day (see AppendixA).

Subgroup v–thiols with oxidised side-chains

At their 53rd, JECFA (2000) allocated the tertiary monothiols to structural class I [FL-no: 12.137, 12.138, 12.145 and 12.252] and structural class II [FL-no: 12.038, 12.085 and 12.259], according to the decision tree approach presented Cramer et al. (1987). JECFA decided that these substances cannot be anticipated to be converted to innocuous metabolites. Therefore, their evaluation proceeded along the B-side of the JECFA procedure. JECFA evaluated these ﬂavouring substances by comparison of their MSDI exposure estimates with the NOAELs of the secondary thiol 2-mercapto-3-butanol ([FL- no: 12.024], JECFA-no: 546) for [FL-no: 12.038, 12.085, 12.137, 12.138, 12.145, 12.252 and 12.259]

and of the secondary thiol cyclopentanethiol ([FL-no: 12.029], JECFA-no: 546) for [FL-no: 12.085].

Adequate margins of safety could be derived and therefore JECFA concluded, at step B4 of the Procedure, that the candidate ﬂavouring substances [FL-no: 12.137, 12.138, 12.145, 12.252, 12.038, 12.085 and 12.259] would not pose a safety concern at their estimated levels of exposure based on the MSDI approach.

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At their 61st meeting, JECFA (2004) allocated the candidate substances [FL-no: 12.169 and 12.241]

to structural class I, according to Cramer et al. (1987) and decided that these two thiols should be evaluated along the B-side of the procedure as no anticipated conversion into innocuous metabolites is expected. JECFA calculated adequate margins of safety by comparing exposure estimate (based on MSDI approach) of [FL-no: 12.169] with a NOAEL for the secondary thiol 3-mercapto-2-pentanone ([FL-no: 12.031], JECFA-no: 560) and exposure estimate of [FL-no: 12.241] with a NOAEL for the secondary thiol 2-mercapto-3-butanol ([FL-no: 12.024], JECFA-no: 546).

At their 76th meeting, JECFA (2012) allocated the candidate substance [FL-no: 12.304] to structural class I, according to Cramer et al. (1987) and decided to evaluate this along the B-side of the procedure as no anticipated conversion into innocuous metabolites is expected. JECFA calculated adequate margins of safety by comparing exposure estimate of [FL-no: 12.304] with NOAELs for the secondary thiol 2-mercapto-3-butanol ([FL-no: 12.024], JECFA-no: 546), a-methyl-b-hydroxypropyl a- methyl-b-mercaptopropyl sulﬁde (JECFA-no: 547) and 3-mercapto-2-pentanone ([FL-no: 12.031], JECFA-no: 560).

Therefore, JECFA concluded that ﬂavouring substances [FL-no: 12.169, 12.241 and 12.304] would not pose a safety concern.

The JECFA safety evaluations ofﬂavouring substances in FGE.91Rev3 are summarised in Table D.1– Appendix D.

EFSA considerations

The FAF Panel disagrees with JECFA with respect to the allocation of the two sulﬁdes [FL-no:

12.065 and 12.079] to structural class I. According to the structural analysis using the OECD (Q)SAR Toolbox (version 4.3.1), the Panel assigns the substances to structural class III.

In line with JECFA, the Panel considers that the two ﬂavouring substances are not expected to be metabolised to innocuous products (step 2) and accordingly evaluates these substances along the B- side of the Procedure.

The estimated daily intake, based on MSDI approach, of the two substances is below the TTC for their structural class III (step B3). The Panel considers that the available NOAEL on the structurally related substance dimethyl sulﬁde ([FL-no: 12.006]) is suitable for a further evaluation of these substances (see EFSA considerations in section 3.3.3.2). Comparison of the MSDIs of [FL-no: 12.065 and 12.079] with the NOAEL of the 90-day study with dimethyl sulﬁde (250 mg/kg bw per day) provides adequate margins of safety (12.5 910⁸and 6.25 910⁸, respectively) for both substances.

Therefore, the Panel concluded, at step B4 of the Procedure scheme, that these two sulﬁdes [FL- no: 12.065 and 12.079] do not pose a safety concern when used as ﬂavouring substances at the estimated levels of intake, based on MSDI approach.

The FAF Panel allocated the tertiary monothiol [FL-no: 12.085] to structural class I, whereas the remaining monothiols [FL-no: 12.038, 12.085, 12.137, 12.138, 12.145, 12.169, 12.241, 12.252, 12.259 and 12.304] to structural class III, in accordance to the structural analysis using the OECD (Q)SAR Toolbox (version 4.3.1).

The Panel agrees with JECFA that these substances cannot be anticipated to be converted into innocuous metabolites, and therefore these substances will be evaluated along the B-side of the Procedure. The MSDIs of all tertiary monothiols are below the TTC for their structural classes (step B3). The Panel does not make use of the NOAELs, derived from secondary thiols, selected by JECFA for the ﬁnalisation of the safety evaluations of the 10 tertiary monothiols [FL-no: 12.038, 12.085, 12.137, 12.138, 12.169, 12.145, 12.241, 12.252, 12.259 and 12.304]. With respect to these 10 substances, the Panel considered that the newly available 90-day toxicity study on 2-methyl-4- oxopentane-2-thiol (4-mercapto-4-methyl-2-pentanone) is suitable to identify a NOAEL of 260 lg/kg bw per day (the highest dose tested). The Panel noted that this 90-day toxicity study has been performed with a test material that did not contain the secondary component 4-methyl-3-penten-2- one, differently from the material of commerce of [FL-no: 12.169] which contains 48–50% of this compound. However, 4-methyl-3-penten-2-one has been evaluated as of no safety concern by EFSA in FGE.63Rev2⁸ (EFSA CEF Panel, 2013) and it is an authorisedﬂavouring substance in the EU Union List under [FL-no: 07.101]. Therefore, the Panel considers the NOAEL of this study with 2-methyl-4- oxopentane-2-thiol (4-mercapto-4-methyl-2-pentanone) suitable for the evaluation of the 10 tertiary

8 The genotoxicity concern for [FL-no: 07.101] was ruled out in FGE.204 (EFSA CEF Panel, 2012b) and then [FL-no: 07.101] was concluded at step A3 of the Procedure as of no safety concern at the estimated levels of intake asﬂavouring substance, based on the MSDI approach, in FGE.63Rev2 (EFSA CEF Panel, 2013).

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monothiols in FGE.91Rev3. With this NOAEL, adequate margins of safety (> 400) for these 10 tertiary monothiols [FL-no: 12.038, 12.085, 12.137, 12.138, 12.169, 12.145, 12.241, 12.252, 12.259 and 12.304] have been calculated. Therefore, the FAF Panel concludes at step B4 of the Procedure scheme that none of these 10 substances would raise a safety concern when used asﬂavouring substances at their estimated levels of exposure based on the MSDI approach.

The stepwise evaluations of allﬂavouring substances in FGE.91Rev3 are summarised in TableD.1 – Appendix D.

4. Discussion

This revision 3 of FGE.91 comprises in total 49 substances, 44 of which had already been considered before in FGE.91Rev2. Two additional ﬂavouring substances [FL-no: 12.065 and 12.079] have been included in this revision, following an extensive evaluation in FGE.201Rev2 of their possible genotoxic potential due to a structural alert for genotoxicity (i.e. a,b-unsaturated carbonyl compounds or precursors for that). Three additional substances [FL-no: 12.169, 12.241] from FGE.74Rev4 and [FL-no:

12.304] from FGE.08Rev5 have been included in the present revision 3 of FGE.91 forﬁnalisation of their safety evaluation. The Panel considered these substances sufﬁciently structurally related to the other tertiary monothiols in this FGE and thus agreed to conclude their evaluation directly in the same FGE.

Because the concern for genotoxicity was ruled out and based on consideration of structural class, metabolism and toxicological data and the MSDI exposure estimates, the FAF Panel concludes that the ﬂavouring substances considered in this revision of FGE.91 (FGE.91Rev3) do not raise a safety concern at step B4 of the Procedure scheme as a NOAEL which provides an adequate margin of safety for all candidate substances exists.

For 44 substances, including the newly added flavouring substances ([FL-no: 12.065, 12.079, 12.169, 12.241 and 12.304]) and the seven tertiary monothiols from FGE.91Rev2 ([FL-no: 12.038, 12.085, 12.137, 12.138, 12.145, 12.252 and 12.259]), normal and maximum use levels have been provided from which mTAMDI exposure estimates have been calculated. The mTAMDI intake estimates for flavouring substances [FL-no: 12.085, 12.137, 12.138, 12.145, 12.169 and 12.241] are below the TTC for their structural classes (I and III). The mTAMDI figures for five substances [FL-no: 12.264, 12.284, 12.274, 12.108, 12.139] from the previous revision (FGE.91Rev2) and five substances in the current revision [FL-no: 12.065, 12.079 12.038, 12.252 and 12.304] are above the TTC for their structural classes. No normal and maximum use levels have been provided for flavouring substances [FL-no: 12.077, 12.162, 12.265, 12.267 and 17.036], previously evaluated in FGE.91Rev2. Therefore, for these 15 substances (more detailed), data on uses and use levels are needed to refine their exposure assessment. On the basis of such data, these flavouring substances should be reconsidered using the Procedure. In order to determine whether the conclusion for the 49 JECFA-evaluated substances can be applied to the materials of commerce, it is necessary to consider the available specifications. Adequate specifications, including complete purity criteria and identity data, are available for all 49flavouring substances.

5. Conclusions

The Panel concludes that for 49 flavouring substances in FGE.91Rev3 there is no safety concern at the estimated levels of intake as flavouring substances, based on the MSDI approach. For 10 flavouring substances [FL-no: 12.065, 12.038, 12.079, 12.108, 12.139, 12.264, 12.274, 12.252, 12.284 and 12.304], the mTAMDI value estimates are above the TTC for their structural classes. Therefore, for these 10 substances more detailed data on uses and use levels are needed in order to refine the exposure assessment and to finalise their safety evaluations. No normal and maximum use levels have been provided for flavouring substances [FL-no: 12.077, 12.162, 12.265, 12.267 and 17.036] which preclude the calculation of their mTAMDI values for comparison with the TTC.

6. Recommendations

The Panel recommends the European Commission to consider:

•

to change the chemical name of [FL-no: 12.169] from 2-methyl-4-oxopentane-2-thiol to 4- mercapto-4-methyl-2-pentanone as indicated in Table B.1–Appendix B;

•

to request normal and maximum use levels for ﬂavouring substances [FL-no: 12.077, 12.162, 12.265, 12.267 and 17.036].

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•

to request more detailed data on uses and use levels forflavouring substances [FL-no: 12.065, 12.038, 12.079, 12.108, 12.139, 12.264, 12.274, 12.252, 12.284 and 12.304] in order to refine the exposure assessment and to finalise their safety evaluations.

7. Documentation provided to EFSA

1) EFFA (European Flavour Association), Submission of additional information on isomeric composition of substances of FGE.91Rev3 ([FL-no: 12.065 and 12.079]).

2) IOFI (International Organization of the Flavor Industry), 1995b. European inquiry on volume of use. IOFI, International Organization of the Flavor Industry, 1995.

3) EFFA (European Flavour Association), 2018d. EFFA 2018 use levels for 63 ﬂavouring substances from FGE.67, 76, 91, 201, 204, 212, 213, 215, 216 and 222. Unpublished data submitted from EFFA to DG SANTE. Dated December 2018.

4) EFFA (European Flavour Association), 2020a. Flavouring Substances-thiols-FGE91_poundages

& Reﬁned-UL_upd 030320. Updated information on nine substances to be evaluated in FGE.91Rev3. Unpublished data submitted by EFFA to EFSA, dated 03/03/2020.

5) EFFA (European Flavour Association), 2017. Submission of Footnote-10 Dossier (“Thiols”):

toxicological studies on Repr. Material: 2-Methyl-4-oxopentane2-thiol [FL-no: 12.169]

(FGE.74Rev3 & FGE.91Rev2) and tonnage data – Footnote-10 substances. Unpublished data submitted by EFFA to EFSA. Dated 12/12/17.

6) M Lloyd BSs, 2014. Induction of micronuclei in cultured human peripheral blood lymphocytes.

2-Mercapto-4-methylpentan-2-one. Covance Laboratories Ltd. Study no. 8261929. May 2014.

Unpublished report submitted by EFFA to EFSA.

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Scientiﬁc opinion on Flavouring Group Evaluation 63 Revision 2 (FGE.63Rev2): consideration of aliphatic secondary alcohols, ketones and related esters evaluated by JECFA (59th and 69th meetings) structurally related to saturated and unsaturated aliphatic secondary alcohols, ketones and esters of secondary alcohols and saturated linear or branched-chain carboxylic acids evaluated by EFSA in FGE.07Rev4. EFSA Journal 2013;11(4):3188, 45 pp.https://doi.org/10.2903/j.efsa.2013.3188

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Scientiﬁc opinion on Flavouring Group Evaluation 91 Revision 2 (FGE.91Rev2): consideration of simple aliphatic and aromatic sulphides and thiols evaluated by the JECFA (53rd and 68th meetings) structurally related to aliphatic and alicyclic mono-, di-, tri-, and polysulphides with or without additional oxygenated functional groups evaluated by EFSA in FGE.08Rev5 (2012). EFSA Journal 2014;12(6):3707, 77 pp. https://doi.org/10.

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Abbreviations

ADME absorption, distribution, metabolism and excretion

bw body weight

CEF EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids DMSO dimethyl sulfoxide

EFFA European Flavour Association

FAF EFSA Panel on Food Additives and Flavourings

FAO Food and Agriculture Organization of the United Nations FEMA Flavor and Extract Manufacturers Association

FGE Flavouring Group Evaluation FL-no FLAVIS number

FLAVIS Flavour Information System (database) GSH glutathione

IOFI International Organization of the Flavor Industry JECFA Joint FAO/WHO Expert Committee on Food Additives MMC mitomycin C

MN micronucleus

MSDI Maximised Survey-derived Daily Intake

mTAMDI modiﬁed Theoretical Added Maximum Daily Intake NOAEL no observed adverse effect level

OECD Organisation for Economic Co-operation and Development SCF Scientiﬁc Committee on Food

TTC threshold of toxicological concern VIN vinblastine

WHO World Health Organization

Scientific Opinion on Flavouring Group Evaluation 91,Revision 3 (FGE.91Rev3): consideration of aliphatic, aromatic and a,b-unsaturated sulfides and thiols evaluated by JECFA(53rd, 61st, 68th and 76th meetings), structurally related to substances in FGE.08

Scienti ﬁ c Opinion on Flavouring Group Evaluation 91, Revision 3 (FGE.91Rev3): consideration of aliphatic, aromatic

and a , b -unsaturated sul ﬁ des and thiols evaluated by JECFA (53rd, 61st, 68th and 76th meetings), structurally related to

substances in FGE.08Rev5

Abstract

Table of contents

1. Introduction

1.1. Background and Terms of Reference as provided by the requestor

1.2. Interpretation of the Terms of Reference

2. Data and methodologies

2.1. Data

2.2. Methodologies

3. Assessment

3.1. Speci ﬁ cations

3.2. Estimation of intake

3.3. Biological and toxicological data

3.4. Application of the Procedure

4. Discussion

5. Conclusions

6. Recommendations

•

•

•

7. Documentation provided to EFSA

References

Abbreviations